Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

Abstract

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD + leads to the suggestion that the carboxylate group of Asp 69 forms a bifurcated hydrogen bond with the 2′ and 3′ hydroxyl groups of the adenosine of NAD + and contributes to the 400-fold preference of the enzyme for NAD + as compared to NADP +. Accordingly, the enzyme with the Asp 69 → Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu 68 → Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2′-phosphate of NADP +. The Glu 68 → Lys:Asp 69 → Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.